Click-ready iridium(<sc>iii</sc>) complexes as versatile bioimaging probes for bioorthogonal metabolic labeling

Herein, we report the synthesis, photophysical characterization and validation of iridium(III)–polypyridine complexes functionalized for click chemistry and bioorthogonal chemistry, as well as their versatile applications as probes in bioimaging studies exploiting metabolic labeling. The designed dyes are conjugated to chemical reporters in a specific manner within cells by CuAAC ligation and display attractive photophysical properties in the UV-visible range. They are indeed highly photostable and emit in the far-red to near-IR region with long lifetimes and large Stokes shifts. We demonstrate that they can be efficiently used to monitor nascent intracellular sialylated glycoconjugates in bioorthogonal MOE studies with a varied panel of optical and non-optical techniques, namely conventional UV-vis laser scanning confocal microscopy (for routine purposes), UV-vis time-resolved luminescence imaging (for specificity and facilitated multiplexing with nano-environment sensitivity), synchrotron radiation based X-ray fluorescence nanoimaging (for high resolution, elemental mapping and quantification in situ) and inductively coupled plasma mass spectrometry (for routine quantification on cell populations with high statistical confidence). The synthesized Ir(III) complexes were utilized in single labeling experiments, as well as in dual click-labeling experiments utilizing two distinct monosaccharide reporters relevant to the same metabolic pathway.